If doing amplicons, please use a lot of phiX or shotgun library (up to 20%) (esp. if fist 25 bp's are the same). It would not hurt, and gains in data quality/yield from better dephasing calculation far outweigh the losses from 5%-20% phiX sequence spike in.

If doing cDNA amplicon analysis: make sure you do not use oligoC oligos in your amplicons, because the read quality would go through the floor if there are 10-14C's or G's in a row, and you may also get non-specific amplification of rRNA (rcDNA), reducing usable data yield by 1-2 orders of magnitude.

Also lower the loading density for 500-600bp amplicons, if you need high quality reads for your analysis.

Obviously do not forget, that the longer the amplicon or the read is, the more sensitive it becomes to the DNA sequence content.

It was an amplicon run, but the cluster density was good (~600K) with 20% phiX, so I expected the Q30 to be much better than 64% overall. It was only 54% for Read 2. Both reads only got out to about 150 bp before dropping off dramatically. The investigator decided it was definitely not worth trying to use the 600-cycle kit again!

It was an amplicon run, but the cluster density was good (~600K) with 20% phiX, so I expected the Q30 to be much better than 64% overall. It was only 54% for Read 2. Both reads only got out to about 150 bp before dropping off dramatically. The investigator decided it was definitely not worth trying to use the 600-cycle kit again!

Oh dear! That's unfortunate, sorry to hear that.

Anyone else with recent experience running the 'new' 600 bp kits? We plan to use one this week or next....

It was an amplicon run, but the cluster density was good (~600K) with 20% phiX, so I expected the Q30 to be much better than 64% overall. It was only 54% for Read 2. Both reads only got out to about 150 bp before dropping off dramatically. The investigator decided it was definitely not worth trying to use the 600-cycle kit again!

Am curious about the focus on the Q-scores. Is the investigator looking to call SNPs? Aligning to reference sequence should be no problem irrespective what the Q-score is.

It was an amplicon run, but the cluster density was good (~600K) with 20% phiX, so I expected the Q30 to be much better than 64% overall. It was only 54% for Read 2. Both reads only got out to about 150 bp before dropping off dramatically.

This looks like issues with adapter dimers to me - what was yours beads ampure cleanup ratios, and any slight bump up on the agilent trace in the region of 100-200bp?

Basically significant amount of the clusters on the run contained shorter templates, and once the end of these is reached (140-160 bp), it wrecks RTA dephasing calculations.

since templates in the 150-200 bp cluster very efficiently compared to 500-800 bp ones, it takes only 3-5% of the shorter contaminant to wreck Q scores in the run.

To minimize it avoid repetitive freeze thaw cycles of the Illumina adapters (they can lose T tails and self ligate) and make sure no nucleases contamination is present during the ligation.

It was an amplicon run, but the cluster density was good (~600K) with 20% phiX, so I expected the Q30 to be much better than 64% overall. It was only 54% for Read 2. Both reads only got out to about 150 bp before dropping off dramatically. The investigator decided it was definitely not worth trying to use the 600-cycle kit again!

Tech support has told me not to cluster below ~750k even for v2 even for amplicon runs. If it's 16S, I had a particular set of samples from some sort of bird guts that I basically can't run by themselves even with 20% phiX because R2 is so bad. There seems to be something happening between cycles 10-30 of R2 that is actually sequence dependent. The way my facility is set up, I mix and match small projects on runs so this is addressable by splitting up the bird samples across many runs.

The suggestion that there may be a primer diamer issue is reasonable, but you also may need to up your density a bit.

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Microbial ecologist, running a sequencing core. I have lots of strong opinions on how to survey communities, pretty sure some are even correct.

It was an amplicon run, but the cluster density was good (~600K) with 20% phiX, so I expected the Q30 to be much better than 64% overall. It was only 54% for Read 2. Both reads only got out to about 150 bp before dropping off dramatically. The investigator decided it was definitely not worth trying to use the 600-cycle kit again!

In addition to above comments it could be a bad batch as well. My current runs Q30 are above %70 specified by Illumina for 2x300 reads:

So.. no this issue is not resolved. I have an email from Illumina today which I will summarise.

The issue is that there is a byproduct in their manufacturing process of fluorescent nucleotides. This byproduct results in NON-CLEAVABLE NTPs - so they limit strand growth. Obviously this has limited effects on the number of clusters per cycle, but does become more acute on 600 cycle kits as the problem 'builds'. You get a lower cluster density, and lower Q30 and higher error rates on the later cycles.

Illumina can now *detect* this byproduct and know what levels impact performance, which sounds to me like there might be batch to batch variation, and have put in place measures to screen for it, therefore they are still expecting improvements 'in the near future' as newer material hits the manufacture pipeline. I am also told there will be a new announcement soon, with new timelines for resolution.